Page 200 - Analytical Electrochemistry 2d Ed - Jospeh Wang

6-1 ELECTROCHEMICAL BIOSENSORS 185

electrochemically. Other types of immunosensors based on labeling the antigen or
antibody with an electroactive tag (e.g., a heavy metal), label-free capacitance
measurements, immobilizing antigen±carrier conjugates at the tip of potentiometric
electrodes, or amplifying the antigen±antibody complex equilibrium by liposome
lysis are also being explored.
Instead of immobilizing the antibody onto the transducer, it is possible to use a
bare (amperometric or potentiometric) electrode for probing enzyme immunoassay
reactions (42). In this case, the content of the immunoassay reaction vessel is
injected to an appropriate ¯ow system containing an electrochemical detector, or the
electrode can be inserted into the reaction vessel. Remarkably low (femtomolar)
detection limits have been reported in connection with the use of the alkaline
phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters
to liberate easily oxidizable phenolic products.

6-1.2.2 DNA Hybridization Biosensors Nucleic acid recognition layers can
be combined with electrochemical transducers to form new and important types of
af®nity biosensors. The use of nucleic acid recognition layers represents a relatively
new and exciting area in biosensor technology. In particular, DNA hybridization
biosensors offer considerable promise for obtaining sequence-speci®c information in
a simpler, faster and cheaper manner, compared to traditional hybridization assays.
Such new strategies hold enormous potential for clinical diagnosis of genetic or
infectious diseases, for the detection of food-contaminating organisms, for environ-
mental monitoring, or for criminal investigations.
The basis for these devices is the Watson±Crick DNA base pairing. Accordingly,
these sensors rely on the immobilization of a relatively short (20±40 bases) single-
stranded DNA sequence (the ``probe'') on the transducer surface, which upon
hybridization to a speci®c complementary region of the target DNA gives rises to
an electrical signal. Several studies have demonstrated the utility of electroactive
indicators for monitoring the hybridization event (45). Such redox-active compounds
have a much larger af®nity for the resulting target:probe duplex (compared to their
af®nity for the probe alone). Their association with the surface duplex thus results in
an increased electrochemical response. For example, Figure 6-14 displays the
increased response of the Co(phen) ‡3 indicator as recorded at a probe-coated strip
3
electrode upon increasing the concentration of the E. coli DNA sequence. Control of
the probe immobilization and of the hybridization conditions (e.g., ionic strength,
temperature, time) is crucial for attaining high sensitivity and selectivity (including
the detection of single point mutations). Sequence-speci®c electrochemical biosen-
sors based on new innovative detection strategies are being developed for direct
electrical detection of the hybridization event. These include the use of enzyme
labels or indicator-free measurements (relying on the intrinsic electroactivity of
DNA, on changes in interfacial properties, or on the conductivity of a DNA-
functionalized electropolymerized ®lm). Ultimately, these developments will lead to
the introduction of miniaturized (on-chip) sensor arrays, containing numerous
microelectrodes (each coated with a different oligonucleotide probe) for the
simultaneous hybridization detection of multiple DNA sequences. The new gene